RNeasy Micro Kit

For purification of up to 45 µg total RNA from cell and tissue samples

Fast procedure delivering high-quality total RNA in minutes

Ready-to-use RNA for high performance in any downstream application

Consistent RNA yields from very small amounts of starting material

No phenol/chloroform extraction

No CsCl gradients, no LiCl or ethanol precipitation

The RNeasy Micro Kit is designed for purification of up to 45 μg RNA from small cell and tissue samples. Tissue samples can be conveniently stabilized using RNAlater RNA Stabilization Reagent or Allprotect Tissue Reagent, and efficiently disrupted using a TissueRuptor or TissueLyser system. RNA purification using the RNeasy Micro Kit can be fully automated on the QIAcube Connect. For larger samples, the RNeasy Mini Kit (spin-column binding capacity of 100 µg RNA), RNeasy Midi Kit (spin-column binding capacity of 1 mg RNA), and RNeasy Maxi Kit (spin-column binding capacity of 6 mg RNA) are also available.

Total RNA was isolated by 5 different users in duplicate from 2 mg of the indicated rat tissues, using the RNeasy Micro Kit. Quantitative, real-time RT-PCR was carried out on an ABI Sequence Detection System using the QuantiTect Probe RT-PCR Kit, with primers and probes specific for c-jun.

High-quality total RNA from fine needle aspirates.

A fine needle aspirate was obtained from rabbit liver using a 19.5-gauge biopsy needle, and the sample was immediately stabilized in an RNAlater TissueProtect Tube. Total RNA was isolated using the RNeasy Micro Kit. The high quality of the RNA is shown by scanning with the Agilent 2100 Bioanalyzer.

RNeasy Micro procedure.

The RNeasy Micro Kit is designed for isolation of total RNA from small samples. Optimized protocols are provided for RNA isolation from microdissected tissues, small amounts of standard tissues, fiber-rich tissues, and cells. Protocols are also available for RNA cleanup and concentration.

Efficient on-column removal of genomic DNA.

Total RNA was isolated from the indicated number of HeLa cells using the RNeasy Micro Kit. Quantitative, real-time PCR was carried out on an ABI Sequence Detection System using the QuantiTect Probe PCR Kit, with primers and probes specific for c-jun. In order to evaluate the efficiency of the on-column DNase digestion, each sample was amplified after reverse transcription (+RT) and directly, without a reverse transcription step (-RT). The lack of signal in the "-RT" samples indicates that no DNA was detectable. Of the "-RT" results, only the 50,000 cell data points are visible.

Reliable RNA isolation from a single cell.

Total RNA was isolated from the indicated number of HeLa cells using the RNeasy Micro Kit. [A] Amplification plot. [B] Correlation of crossing point (CP) values and cell numbers. Real-time RT-PCR was carried out on the LightCycler System using the QuantiTect Probe RT-PCR Kit, with primers and probes specific for β-actin.

The RNeasy Micro Kit is designed for isolation of total RNA from small samples such as microdissected tissues and fine needle aspirates, from milligram amounts of fibrous tissues including heart and muscle tissue, and from small numbers of cells down to single cells (e.g., FACS sorted cells). For microdissected FFPE tissues, we recommend the RNeasy FFPE Kit. RNeasy technology simplifies total RNA isolation by combining the stringency of guanidine-isothiocyanate lysis with the speed and purity of silica-membrane purification and the unique design of RNeasy MinElute spin columns enables minimal elution volumes, as little as 10 µl, to concentrate the RNA. The small elution volume means that reaction volumes can be kept small in downstream applications, giving increased reaction efficiency. The high purity allows the complete sample to be used in downstream reactions, such as reverse transcription, without enzyme inhibition.

Procedure

The RNeasy Micro Kit is designed for isolation of total RNA from small samples. Samples (<5 mg tissue, or <5 x 105 cells) are first lysed and then homogenized. Ethanol is added to the lysate to provide ideal binding conditions. The lysate is then loaded onto the RNeasy MinElute spin column and RNA (up to 45 µg) binds to the silica membrane. RNase-free DNase, provided in the RNeasy Micro Kit, enables simple and efficient on-column digestion of genomic DNA for sensitive applications. Then DNase and any contaminants are efficiently washed away, and pure, concentrated RNA is eluted in 10–14 µl water.

Applications

Highly reproducible isolation of RNA in a small elution volume (10 µl) using the RNeasy Micro Kit makes the highly pure RNA well-suited for sensitive quantitative gene expression analyses, including real-time RT-PCR starting with as little as one cell.